Int.J.Curr.Microbiol.App.Sci (2018) 7(11): 2829-2843

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 11 (2018) Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2018.711.326

Conventional and Molecular Detection of Listeria monocytogenes and its Antibiotic Sensitivity Profile from Cattle Sources of Aizawl, Mizoram (India)

Papia Biswas1*, Devajani Deka1, T.K. Dutta2, E. Motina1 and P. Roychoudhury2

1Department of Veterinary Public Health & Epidemiology, 2Department of Veterinary Microbiology, College of Veterinary Sciences & AH, Central Agricultural University, Selesih, Aizawl, Mizoram, 796014, India

*Corresponding author

ABSTRACT

The present study was conducted to study the prevalence of the food borne zoonotic pathogen of animal origin, L. monocytogenes by and identification, molecular detection and antibiotic sensitivity pattern from different samples of cattle sources in Aizawl district of Mizoram. A total 200 numbers of sample including cattle faeces (50), K e yw or ds raw milk (50) and milk products (100) were collected randomly from different unorganized shop and farms. The seasonal variation in the occurrence of L. monocytogenes Listeria monocytogenes, PCR, Antibiotic was also studied. The L. monocytogenes was isolated by using two step enrichment method sensitivity, Aizawl, of culturing and identified based on cultural characteristics, gram , biochemical Mizoram properties, tumbling motility and in vitro pathogenicity tests. The molecular detection of L. Article Info monocytogenes strains were done by PCR using published primers. The antibiotic sensitivity was studied against 12 numbers of commonly used antibiotics in animals and Accepted: human. The prevalence of L. monocytogenes was recorded as 6.50 percent including 8.00 22 October 2018 percent from cattle faeces, 6.00 percent from raw milk, 8.00 percent from lassi, dahi and Available Online: 10 November 2018 ice-cream samples, respectively. The L. monocytogenes strains showed 100 percent sensitivity towards Penicillin, Ampicillin, Oxacillin, Cephotaxime/Clavulanic acid,

Ciprofloxacin, Tetracycline and Trimethoprim/Sulphamethoxazole followed by Streptomycin (84.61%), Chloramphenicol (53.84%), Gentamicin (53.84%) and Ceftriaxone (46.15%). Introduction monocytogenes is the most important species in the genus can be secreted through milk of Listeriosis has been among important food both healthy and infected animals (Wagner et borne zoonotic diseases since long, mostly due al., 2000). This is still called an emerging to its high mortality rate despite of being pathogen as its transmission through uncommon in human beings (Atil et al., contaminated food is recently recognized. L. 2011). The severity of the disease has become monocytogenes is a gram positive, ubiquitous, significant as the causative organism Listeria non-spore forming organism that can survive a 2829

Int.J.Curr.Microbiol.App.Sci (2018) 7(11): 2829-2843 wide range of pH from 4.0-9.6 and Period of the study temperature from -1.5°C to 45°C (Lado and Yousef, 2007). Listeriosis is commonly The study was conducted for a period of one characterized by meningoencephalitis, year from July, 2017 to June, 2018 and the generalised septicaemia and abortion (Late study period was divided into two halves; pregnancy) in both human and animals. Summer (March to September) and Winter Young individuals along with (October to February). immunocompromised ones are more susceptible than others. Way back in 1985, Collection of Samples Listeriosis was declared as serious public health hazards (Rocourt and Catimel, 1985). A total of 200 numbers of faecal samples of cattle, raw milk and milk products were Listeria spp. have been reported as susceptible collected randomly from different unorganized to antibiotics active against gram positive cattle farms/ milk vendors/ shops periodically bacteria but in recent years like many other during the study period by following aseptic bacterial pathogens Listeria are developing measures for detection of L. monocytogenes resistance to many currently used antibiotics. during the study period. Distributions of Current choice of antibiotics for all forms of different samples collected are given in the listeriosis is combination of Ampicillin and Table 1. Gentamicin (Schlech and Acheson, 2000). The studies on L. monocytogenes in the Isolation and phenotypic characterization perspective of foodborne pathogen are scanty of L. monocytogenes in North-East Region states of India including Mizoram. Therefore keeping the above points Enrichment of faecal sample in view, the present study was undertaken to isolate, identify and to study the prevalence The USDA (USDA FSIS, 2002) method was and antimicrobial sensitivity pattern of L. employed for isolation of Listeria spp. from monocytogenes from different samples of faecal samples of cattle by two step cattle source s in Aizawl district of Mizoram. enrichment method. Primary enrichment of five grams of faecal sample was done in 45 ml Materials and Methods 1/2 strength UVM-I broth containing selective supplements (HiMedia Pvt. Ltd., Mumbai)® Study area and incubated for 24 hours at 30°C followed by secondary enrichment of 0.1 ml from the The present study on isolation and primary broth culture in 10 ml UVM-II broth identification, molecular detection and containing selective supplements and antimicrobial sensitivity pattern of L. incubated for 48 hours at 37°C. monocytogenes from different samples of cattle sources was carried out in Aizawl Enrichment of milk and milk products district of Mizoram. It is mainly a hilly state of North-eastern region. It extends from 21°56'N Food and Drug Administration (2015) testing to 24°31'N, and 92°16'E to 93°26'E. It is methodology with slight modification was the 2nd least populous state in the country and employed for isolation and identification of L. it is covers an area of approximately 21,087 monocytogenes from cattle faeces, raw milk square kilometres. About 91percent of the area and milk products (lassi, dahi, ice-cream and in the state is forested. rasmalai). Twenty five ml of sample was

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Int.J.Curr.Microbiol.App.Sci (2018) 7(11): 2829-2843 mixed with 225 ml of UVM broth properly for surrounded by a small zone of β-haemolysis 2 minutes and the mixture was incubated at 30 after back light. ± 2°C for 24 hours. For secondary enrichment, 0.1ml of the cultured UVM was transferred to Christie, Atkins, Munch- Petersen (CAMP) 10 ml of Fraser broth (FB) and incubated at Test 37°C for 24 ± 2 hours. The presence of in-vitro pathogenicity of L. Selective plating of L. monocytogenes monocytogenes by CAMP test was as per the (UVM-Broth and FB culture) in PALCAM, method of ISO (1996). The standard strains of McBride and TSYEA agar Rhodococcus equi (MTCC 8144) and (MTCC 43300) were A drop of approximately 0.1ml of FB broth streaked on freshly prepared 5 percent SBA culture turning to black colour was streaked plates wide apart and parallel to each other. aseptically upon PALCAM and McBride agar The test strains were streaked at 90° angle to plates and the plates were incubated at 37°C R. equi and S. aureus with a distance of three for 24–48 hours. The suspected colonies on mm apart from these strains line. PALCAM/ McBride agar plates were streaked with the help of a sterile loop on TSYEA plate The streaked plates were incubated for 24 and incubated at 37°C for 24 hours and hours at 37°C and examined for haemolytic subsequently tested for further biochemical zone from partial haemolysis to a wider zone and in vitro pathogenicity characteristics. of complete haemolysis. The isolates with CAMP- positivity against S. aureus were Morphological and biochemical characterized as L. monocytogenes giving a characteristics of L. monocytogenes spade shaped haemolytic zone formation.

The L.monocytogenes strains were Molecular detection of L. monocytogenes phenotypically characterized by morphological characteristics, Gram staining Bacterial lysate preparation reaction and biochemical characteristics (, Oxidase, Motility, Indole, Methyl All the culturally, phenotypically and Red, Voges-Proskauer, Citrate utilization, biochemically positive L. monocytogenes fermentation patterns of sugars like L- isolates were processed for bacterial lysate Rhamnose, D-Xylose and Mannitol etc.) preparation using boiling and snap chill (Quinn et al., 1994). method. A single colony of phenotypically confirmed strain was inoculated into one ml of In vitro pathogenicity test LB broth and incubated at 37°C for 16-18 hours. After overnight incubation at 37°C, Beta haemolysis test on five percent sheep cells were pelleted by centrifugation at 8000 blood agar rpm for 10 minutes at 4°C. Then the pellet was washed three times with sterile normal saline The suspected colonies on PALCAM/ solution (0.85%) and finally re-suspended in McBride/ TSYEA agar plates were streaked 500µl of nuclease free sterile distilled water. on five percent Sheep Blood Agar (SBA) The cell suspension was heated in a boiling plates and the plates were incubated at 37°C water bath for five minutes followed by for 24 hours. The L. monocytogenes positive immediate chilling. The cellular debris was SBA plate showed translucent colonies sediment by centrifugation at 5000 rpm for

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Int.J.Curr.Microbiol.App.Sci (2018) 7(11): 2829-2843 five minutes. The supernatant was used as Penicillin G, Ampicillin, Oxacillin, template DNA for PCR assay. Streptomycin, Erythromycin, Cephotaxime/ Clavulanic acid, Ceftriaxone, Detection of species specific gene (16S- Chloramphenicol, Ciprofloxacin, Gentamicin, rRNA) of L. monocytogenes isolates by PCR Tetracycline and Trimethoprim/ Sulphamethoxazole as per Clinical and All the culturally, phenotypically and Laboratory Standard Institute (CLSI) biochemically positive L. monocytogenes guidelines (2014). The L. monocytogenes isolates were subjected for 16S-rRNA species isolates were inoculated into Brain Heart specific gene amplification by PCR using Infusion (BHI) broth and incubated for 24 published primer and according to the hours at 37°C. After that, 200 µl of each methodology described by Jallewar et al., inoculum was taken on Muller Hinton agar (2007). The details about the primer sequence plates and spread eventually with the help of are given at Table 2. The PCR assay was sterile L-shaped spreader. Then the plates carried out in 0.2 ml thin PCR tube. To detect were allowed to dry and antibiotic discs were species specific genes of L. monocytogenes, placed on media aseptically with the help of the PCR protocol was standardized by using sterile forceps. Next, the plates were incubated standard L. monocytogenes (MTCC 1143) as at 37°C for 24-48 hours. After completion of positive control and sterile milli-Q water as incubation the diameter of zone of inhibition negative control. The final composition for 25 was compared with the standard known value μl reaction mixture is given at Table 3. against each specific antimicrobial agent from interpretation guide line (Hi-Media)®. Amplification of DNA was performed in a Thermal cycler machine with a pre-heated lid. Results and Discussion The detail of the cycling condition for the species specific gene was given in the Table 4. Isolation and identification of L. All the amplified PCR products were analyzed monocytogenes by agarose gel electrophoresis using one percent agarose gel in 1X TAE buffer (pH Out of 200 different samples from cattle 8.0). About five µl of PCR product was mixed sources (cattle faeces, raw milk and milk with 2 μl of 6 X gel loading dye and loaded products) of Aizawl, a total 29 (14.50%) into each well. DNA ladder (3000 bp) was samples were found to be positive for Listeria used as reference to compare the size of spp. by the cultural method in which isolates amplified products. The gel was visualized turned into black colour in different broth (FB under UV transilluminator (Alpha Imager) and and UVM) and also showed different documented by gel documentation system characteristics of colonies on different agars (Alpha Imager). such as green colonies with black haloes in PALCAM agar, dense white to iridescent Detection of antibiotic sensitivity and white appearing as crushed glass in McBride resistance pattern of L. monocytogenes agar and clean glass like colonies in TSYEA strains agar after 24-48 hours of incubation at 37°C. Based on the Gram staining reaction and All the L. monocytogenes isolates were different biochemical tests (catalase: positive:; subjected to in vitro antibiotic sensitivity test oxidase: negative: tumbling motility; indole: by disc diffusion method (Bauer et al., 1966) negative; : positive; Voges- against a panel of 12 antibiotics namely Proskauer: positive citrate: negative; L-

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Int.J.Curr.Microbiol.App.Sci (2018) 7(11): 2829-2843 rhamnose: positive; D- Mannitol fermentation: 8.00 percent (2/25) from lassi, dahi and ice- negative; D- Xylose fermentation: negative, cream samples, respectively (Table 7 and weak haemolysis on sheep blood agar and Figure 13). Listeria monocytogenes was positive CAMP test against Staphylococcus detected from the raw milk and ready to eat aureus characteristics), 13 (6.50%) numbers refrigerated milk products produced locally of L. monocytogenes were identified and the from unpasteurized milk like dahi, lassi and findings were in accordance with Gupta and ice-cream whereas the organism was not Sharma (2012) and Walse et al., (2003) (Table isolated from rasmalai which is a well-cooked 5, 6 and Figure 1–12). milk product stored for a short duration of time in the sweet shops. The Higher The detection of Listeria spp. from food prevalence rates of L. monocytogenes from products is challenging due to the concurrence faecal samples of ruminants were recorded by presence of other organisms within the food Lawan et al., (2003) (10.00%) and Kalorey et product. In this respect, the isolation method al., (2006) (16.00%) from Nigria and Nagpur in respect to specific pathogen is critical and (India), respectively. Waghmare, (2006) must allow recovery and detection of injured evaluated the incidence of Listeria spp. in raw cells too. In food, detection of Listeria spp. is milk from different markets of Mumbai city generally performed in a two-step cultural (India) and revealed prevalence of Listeria enrichment process and along with selective spp. and L. monocytogenes amongst the supplements like antibacterial and antifungal pasteurized milk samples with the incidence of agents. The bacteriological culture methods 21.32 and 5.88 per cent in unpasteurized milk commonly used for detection and samples. Similarly, Chandio et al., (2007) identification of the bacteria include aesculin reported 6.00 per cent of L. monocytogenes in and ferric iron in enrichment or plating media, raw cow milk where as higher incidence of which results through the hydrolysing capacity prevalence of L. monocytogenes (21. 70%) of Listeria spp., in the formation of intense was reported by Sharma et al., (2012) from black colour (Fraser and Sperber, 1988). 115 raw cow milk samples in Meerut and Results of in vitro pathogenicity tests showed Babugarh Cantt, Hapur, India. In contrast, that Listeria spp. brought about haemolysis on studies conducted at Coimbatore (Tamilnadu) five per cent SBA similar to the earlier records and Mangalore, India reported that branded of Blanco et al., (2008). The Christie Atkins milks were more prone to L. monocytogenes Munch-Petersen (CAMP) test is a unique than the local milk (Dhanashree et al., 2003; confirmatory tool for identification of this Sheela and Muthukmar, 2011). However, food borne pathogen. The Listeria spp. Moharram et al., (2007) reported 5.00 percent isolates recovered during the study have incidence of L. monocytogenes from non- shown the positive CAMP pattern against S. branded ice-cream samples from different ice aureus (ISO, 1996). cream parlours of Mysore (India).

Prevalence of L. monocytogenes in different The seasonal distribution of L. monocytogenes samples of cattle sources (faeces, raw milk revealed 4.95 and 8.08 percent of prevalence and milk products) from Aizawl, Mizoram in summer and winter season, respectively. The Seasonal fluctuation of L. monocytogenes The prevalence of L. monocytogenes was in the milk has been reported as 1.69 per cent recorded as 6.50 percent (13/200) comprised in summer and 3.82 per cent in winter (Aurora of 8.00 percent (4/50) strains from cattle et al., 2006) (Table 8 and Figure 14). faeces, 6.00 percent (3/50) from raw milk,

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Table.1 Distribution of different samples of cattle sources collected from Aizawl district of Mizoram

Sl. No State Type of sample Number of samples Seasonal distribution Summer Winter 1 Aizawl Cattle faeces 50 25 25 2 (Mizoram) Raw cow milk 50 25 25 3 Lassi 25 13 12 4 Dahi 25 13 12 5 Ice-cream 25 12 13 6 Rasmalai 25 13 12 Total 200 101 99

Table.2 Oligonucleotide primers used for detection of species specific gene of L. monocytogenes by PCR

Target Primer Sequence (5’-3’) Base Reference Genes Pair (bp) 16Sr- F- 1200 Weidmann RNA GGACCGGGGCTAATACCGAATGATAA et al.,(1993) R- TTCATGTAGGCGAGTTGCAGCCTA

Table.3 Composition of PCR reaction mixture for detection of species specific gene and virulence genes of L. monocytogenes

Sl. No. Ingredients Volume (µl) 1 PCR Master Mixture 2x 12.5 2 Forward primer 1 3 Reverse primer 1 4 Template 4 5 Milli-Q water 6.5 Total 25.0

Table.4 Thermal cycling condition for detection of species specific (16S-rRNA) gene of L. monocytogenes

Sl. No Stages PCR for 16Sr-RNA gene of L. monocytogenes 1 Initial denaturation 94°C for 4 min 2 Denaturation 94°C for 30 sec 3 Annealing 56.5°C 45 sec 4 Elongation 72°C for 30 sec 5 Final Extension for 1 cycle 72°C for 3 min No. of cycle 35

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Table.5 Morphological and biochemical test results of L. monocytogenes

Sl. No. Morphological/biochemical test Positive characteristics 1 Gram staining Positive 2 Catalase Positive 3 Oxidase Negative 4 Motility Tumbling 5 Indole Negative 6 Methyl Red Positive 7 Voges-Proskauer Positive 8 Citrate Negative 9 L-Rhamnose fermentation Positive 10 D- Mannitol fermentation Negative 11 D- Xylose fermentation Negative

Table.6 Listeria monocytogenes isolation by cultural method and confirmed by biochemical test collected from different source of cattle of Aizawl (Mizoram) district

Sl. State Type of Number Number of Number of samples No. sample of samples positive positive for L. samples for Listeria spp. by monocytogenes after analyzed cultural method biochemical test 1 Aizawl Cattle faeces 50 7 4 2 (Mizoram) Raw cow milk 50 9 3 3 Lassi 25 4 2 4 Dahi 25 4 2 5 Ice-cream 25 5 2 6 Rasmalai 25 - - Total 200 29 13

Table.7 Prevalence of L. monocytogenes in different samples of cattle source from Aizawl (Mizoram) district (n=200)

Sl. State Type of Number of Number of % prevalence of No. sample samples sample positive L. analyzed for L. monocytogenes monocytogenes 1 Cattle faeces 50 4 8.00 2 Raw milk 50 3 6.00 3 Aizawl Lassi 25 2 8.00 4 (Mizoram) Dahi 25 2 8.00 5 Ice-cream 25 2 8.00 6 Rasmalai 25 0 0.00 Total 200 13 6.50

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Table.8 Season wise prevalence of L. monocytogenes isolated from different samples of cattle source from Aizawl (Mizoram) district

Sl. State Type of Number Number Seasonal distribution of L. No. sample of sample of sample monocytogenes tested in tested in Prevalence Prevalence in winter winter in summer winter 1 Cattle 25 25 1 (4.00%) 3 (12.00%) faeces Aizawl 2 (Mizoram) Raw milk 25 25 1 (4.00%) 2 (8.00%) 3 Lassi 13 12 1 (7.69%) 1 (8.33%) 4 Dahi 13 12 1 (7.69%) 1 (8.33%)

5 Ice-cream 12 13 1 (8.33%) 1 (7.69%) 6 Rasmalai 13 12 - - Total 101 99 5 (4.95%) 8 (8.08%)

Table.9 Antibiotic Sensitivity and Resistance pattern of L. monocytogenes isolated from different samples of cattle source of Aizawl (Mizoram) district

Sl. Antimicrobial agent No. of L. monocytogenes isolated from cattle faeces No. isolates Sensitive Intermediate Resistance (%) (%) (%) 1 Penicillin G (P) 13 13 100 - - - - 2 Ampicillin (AMP) 13 13 100 - - - - 3 Oxacillin (OX) 13 13 100 - - - - 4 Streptomycin (HLS) 13 11 84.61 2 15.38 - - 5 Erythromycin (E) 13 - - 1 7.69 12 92.30 6 Cephotaxime / 13 13 100 - - - - Clavulanic acid (CEC)

7 Ceftriaxone (CTR) 13 6 46.15 - - 7 53.84 8 Chloramphenicol (C) 13 7 53.84 2 15.38 4 30.76 9 Ciprofloxacin (CIP) 13 13 100 - - - - 10 Gentamicin (GEN) 13 7 53.84 - - 6 46.15 11 Tetracycline (TE) 13 13 100 - - - - 12 Trimethoprim/Sulpha 13 13 100 - - - - methoxazole (COT)

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Figure 2: L. monocytogenes Figure 3: L. monocytogenes on Figure 1 : Milk products with on PALCAM agar McBride agar UVM - I

Figure 4: L. monocytogenes Figure 5: L. monocytogenes Figure 6: L. monocytogenes on TSYEA agar showing gram staining showing oxidase negative and positive Catalase positive

Figure 7: L. Figur e 8: IMVIC test Figure 9: Sugar monocytogenes showing showing MR and VP +ve fermentation tests for L. umbrella shaped growth for L. monocytogenes monocytogenes (Rhamnose +ve, Xylose and Mannitol – in Listeria motility

ve)

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Figure 10: L. monocytogenes showing Figure 11: L. monocytogenes weak ? - haemolysis on SBA showing positivity in CAMP test

0 Ice- cream 2 5 2 4 Lassi 2 4 3 9 Cattle faeces 4 7

Type of sample 0 1 2 3 4 5 6 7 8 9 10 No. of samples

Positive for L.monocytogenes by biochemical tests Positive for Listeria spp. cultural method

Figure 12: Detection of Listeria monocytogenes from different samples of cattle source from Aizawl (Mizoram) district by cultural method (n=200)

Figure - 1 3 Prevalence of L. monocytogenes in different samples of cattle source from Aizawl (Mizoram) district (n=200) s 2838

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15% 12% 8.33% 8.33% 10% 8% 7.69% 7.69% 8.33% 7.69% 4% 4% 5% 0 0 0%

Percentage(%) Cattle faeces Raw milk Lassi (n=25) Dahi (n=25) Ice-Cream Rasmalai (n=25) (n=50) (n=50) (n=25)

Type of sample

Prevalence in Summer Prevalence in Winter

Figure 14: Season wise prevalence of L. monocytogenes isolated from different samples of cattle source from Aizawl (Mizoram) district (n=200)

L1 L2 L3 L4 L5 L6 L7 L8 L9

1500 bP

1000 bp

1200 bp

Figure-15: Agarose gel electrophoresis showing the PCR amplicons of 16S-rRNA gene (1200bp) obtained from L. monocytogenes strains; L1: 3000 bp DNA ladder; L2: Positive control; L3: Negative control; L4 to L9: Representative samples

Figure 16: Antibiotic sensitivity and resistance pattern of L. monocytogenes isolated from different samples of cattle source of Aizawl (Mizoram) district

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Listeria is a widely distributed bacterium in Antibiotic sensitivity pattern of L. nature and commonly found in soil, sewage, monocytogenes dust, water and causes listeriosis in humans and animals (Norton et al., 2001). Of the All the 13 L. monocytogenes strains showed various milk pathogens, L. monocytogenes is 100 percent sensitivity towards Penicillin, one of the deadly organisms which occurs Ampicillin, Oxacillin, Cephotaxime/ largely in all types of environment, including Clavulanic acid, Ciprofloxacin, Tetracycline foods grown in contaminated environment, and Trimethoprim/ Sulphamethoxazole poorly processed/stored food, milk and followed by Streptomycin (84.61%), associated products (Priyanka and Alka, Chloramphenicol (53.84%), Gentamicin 2008). The study of incidence of Listeria spp. (53.84%) and Ceftriaxone (46.15%). in cattle faeces, milk and milk products in Conversely the L. monocytogenes strains their selling units provide information about showed highest resistance to Erythromycin the carrier status in cattle and contamination (92.30%), Ceftriaxone (53.84%), Gentamicin status of the milk and milk products. (46.15%) and Chloramphenicol (30.76%), respectively (Table 9 and Figure 16). The milk producing and processing environment and handling practices may vary There is growing concern of bacterial place to place and production practices. adaptation and evolution resulting in the emergence of antimicrobial resistant bacteria There are chances of increase in cross pathogens since last 50 years. The prevalence contamination as 47 per cent of surface of of antimicrobial resistance among food borne hand of the food handlers and 16 per cent on pathogens has increased during recent the processing tables were found to carry L. decades (Akbar and Anal, 2014). The monocytogenes (Kerr et al., 1993; frequent and unnecessary use of antimicrobial Jayasekaran et al., 1996). The presence of agents in food animals for therapeutic and Listeria spp. particularly L. monocytogenes in prophylactic purposes in animals are ready to eat milk products like dahi, lassi, ice contributing to create resistant strains. Animal cream and raw milk could be a major food origin foods are the major sources of safety issue for consumers as L. transmission of antimicrobial resistant monocytogenes should be absent in RTE organisms to human. The antimicrobial foods (US-FDA) (Fusch et al., 1992). resistant bacteria from food animals may colonize the human population via food chain, Detection of species specific gene (16S- contact through occupational exposure or rRNA) of L. monocytogenes in different waste run off from animal production samples of cattle source facilities. Resistant bacteria may readily transferred from food animals to human The culturally, phenotypically and beings as the similar kind of antimicrobial biochemically positive 13 numbers of L. agents are used in human practice also, monocytogenes isolates were subjected for therefore the detection of antimicrobial 16S-rRNA species specific gene amplification resistance pattern is a matter of public health using the standardized PCR protocol by using significance. Sharma et al., (2012) detected published primer. All the 13 numbers of L. 80-90 percent resistance of L. monocytogenes monocytogenes strains isolated from Aizawl strains from raw milk of Meerut and districts of Mizoram were positive for 16S- Babugarh Cantt, Hapur (India) to Nalidixic rRNA gene (Figure 15). acid, Amoxycillin + Sulbactum, Vancomycin,

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Kanamycin, Cloxacillin, and Erythromicin References whereas many were susceptible to the Ampicillin, Ofloxacin, Tetracycline, Akbar, A., and Anal, A.K. 2014. Zinc oxide Streptomycin, Sulphafurazole, Oxacilin and nanoparticles loaded active packaging a Ciprofloxacin. challenge study against typhimurium and Staphylococcus The findings of Sharma et al., (2017) is aureus ready-to-eat poultry meat. Food alarming as they recently isolated Multi Drug Contr., 38:88-95. Resistant (MDR) strains of L. monocytogenes Atil, E., Ertas, H.B., and Ozbey, G. from raw milk in Rajasthan and emphasized 2011.Isolation and molecular on the need of awareness among consumers. characterization of Listeria spp. From animals, food and environmental Implementation of food safety regulations at samples. Vet. Med., 56: 386 394. different levels of milk production has come Aurora, A., Prakash, A., and Prakash, S. up as a great public health issue. 2006. Occurrence of pathogenic Listeria monocytogenes in raw milk and ready to The present study detected the L. eat milk products in Agra city. India. monocytogenes, a major zoonotic pathogen Indian. J. Comp. Microbiol. Immunol. causing fatal infections in human by Infect. Dis., 27(2): 87-93. conventional and molecular detection Bauer, A.W., Kirby, W.M.M., Sherris, J.C., methods in different samples of cattle sources and Turck, M. 1966. Antibiotic namely faeces, raw milk and milk products in susceptibility testing by a standardized the study area indicating the public health single disc method. Amer. J. Clin. significance of the pathogen. Patho., 45:493-496. Blanco, M. B., Fernandez-Garayzabal, J. F., The presence of the organism in cattle faeces Dominguez, L., Briones, V., Vazquez- indicated the carrier status and the presence in Boland, J. A., Blanco, J. L., Garcia, J. raw milk and refrigerated milk products A., and Suarez, G. 2008. A technique produced locally and sold in local markets for the direct identification of under unhygienic condition is alarming public haemolytic pathogenic Listeria on health threat to the consumers. selective plating media. Lett. Al. Microbiol., 9:125-128. The well-cooked milk product (Rasmalai) Chandio, T.H., Soomro, A.H., Bhutto, M.B., which is stored for a short period of time has Dewani, P., and Shah, G. 2007. been found to be free from L. monocytogenes. Occurrence of Listeria monocytogenes in bovine milk in Hyderabad, Pakistan. Acknowledgements Ann. Microbiol., 57:341- 344. CLSI (2014). Performance Standards for The authors duly acknowledge to the Dean, Antimicrobial Microbial Susceptibility College of Veterinary Sciences & Animal testing 18th Information supplement Husbandry, Central Agricultural University, M100-S18. Wany, PA: Clinical and Selesih, Aizawl, Mizoram for providing the Laboratory Standards Institute. funds with necessary facilities to conduct this Dhanashree, B., Otta, S.K., and Karunasagar, study under the Department of Veterinary I. 2003. Typing of Listeria Public Health and Epidemiology. monocytogenes isolates by random

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amplification of polymorphic DNA. of Listeria spp. on the hands of food Indian J. Med. Res., 117:19–24. workers. J. Food Prot., 56:525-527. FDA 2015. Testing methodology for L. Lado, B., and Yousef, A. E. 2007 monocytogenes in environmental Characteristics of Listeria samples. Version 1. monocyotgenes important to food Fraser, J.A., and Sperber, W.H. 1988. Rapid processors, Chapt. 6. In: Ryser E T and detection of Listeria spp. in food and Marth E H (Eds), Listeria, listeriosis environmental samples by esculin and food safety, 3rd edn, CRC Press hydrolysis. J. Food Prot., 51:762-765. Taylor and Francis Group, Boca Raton. Fusch, R.S., and Reilly, P.J.A. 1992. The Pp. 157-213. incidence and significance of in sea Lawan, F. A., Tijjani, A. N., Raufu, A. I., foods. In: H. H. Huss, and M. Jackobsen Ameh, J. A., Ngoshe, I. Y., and Auwal, (ed), Proceeding of an International M.S. 2003. Isolation and Conference on ―Quality Assurance in characterization of Listeria species from the Fish industry‖, Copenhagen, ruminants in Maiduguri north-eastern Denmark pp. 217-230. Nigeria. Afr. J. Biotechnol., Gupta, S., and Sharma, V. 2012. Antibiotic 12(50):6997-7001. resistance pattern among different Moharram, M., CharithRaj, A.P., and Listeria species isolated from mutton Janardhana, G.R. 2007. Prevalence of and chevon. J. Anim. Res., 3: 99-102. Listeria monocytogenes in ice creams International Organization for sold in Mysore city and detection by Standardization, 1996. Microbiology of Polymerase Chain Reaction (PCR). food and animal feeding stuffs- Asian J. Microbiol. Biotechnol. Horizontal method for detection and Environ. Sci., 9:151-154. enumeration of Listeria monocytogenes- Norton, D.M., McCamey, M.A., Gall, K.L., part-1: Detection method. International Scarlett, J.M., Boor, K.J., and Standard ISO11290-1, Geneva, Wiedmann, M. 2001. Molecular studies Switzerland. on the ecology of Listeria Jallewar, P.K., Kalorey, D.R., Kurkure, N.V., monocytogenes in the smoked fish Pande, V.V., and Barbuddhe, S.B. 2007. processing industry. Appl. Environ. Genotypic characterization of Listeria Microbiol., 67: 198-205. spp. isolated from fresh water fish. Int. Priyanka, S., and Alka, P. 2008. Isolation of J. Food Microbiol., 114: 120–123. , Staphylococcus aureus Jayasekaran, G., Karunasagar, I., and and Listeria monocytogenes from milk Karunasagar, I. 1996. Incidence of products sold under market conditions Listeria species in tropical fish. Int.J. at Agra region. Acta agriculturae Food Microbiol., 31: 333-340. Slovenica., 92: 83–88. Kalorey, D.R., Kurkure, S.R., Rawool, D.B., Quinn, P.J., Carter, M.E., Markey, B.K., and Mallik, S.V.S., and Barbuddhe, S.B. Carter, G.R. 1994. General procedures 2006. Isolation of pathogenic Listeria in microbiology. Clin. Vet. Microbiol. monocytogenes in faeces of wild Wolfe Publishing, London, pp. 648. animals in captivity. Comp. Immunol, Rocourt, J., and Catimel, B. 1985. Microbiol. Inf. Dis., 29:295-300. Biochemical characterization of species Kerr, K.G., Birkenhead, D., Seale, K., Major, in the genus Listeria. Zentralble J., and Hawkey, P.M. 1993 Prevalence Bakteriologika Mikrobiol. Hyg., 260:221 231.

2842

Int.J.Curr.Microbiol.App.Sci (2018) 7(11): 2829-2843

Schlech III, W.F., and Acheson, D. 2000. monocytogenes from Red Meat, Poultry, Foodborne listeriosis. Clin. Infect. Dis., Egg and Environmental Samples, 31(3): 770-775. Revision 03, April 29, 2002. In: Sharma, D., Sharma, P.K., Saharan, B.S., and Microbiol. Lab. Guidebook. pp 1–21. Malik, A. 2012. Isolation, identification Waghmare, R.N. 2006. Prevalence and and antibiotic susceptibility profiling of Molecular characterization of Listeria antimicrobial resistant Listeria monocytogenes from milk. Thesis, monocytogenes from dairy milk. Int. J. M.V.Sc, MAFSU, Bombay Veterinary Microbial. Res. Technol., 1: 1-4. College, Mumbai. Sharma, S., Sharma, V., Dahiya, D.K., Khan, Wagner, M., Podstatzky-Lichtens, L., A., Mathur, M., and Sharma, A. 2017. Lethner, A., Asperger, H., Baumgarther, Prevalence, virulence potential, and W., and Brand, E. 2000. Prolonged antibiotic susceptibility profile of excretion of Listeria monocytogenes in Listeria monocytogenes isolated from a subclinical case of mastitis. bovine raw milk samples obtained from Milchwisschaft, 55: 3 6. Rajasthan, India. Foodborne. Pathog. Walse, S.H., Paturkar, A.M., Sherikar, A.T., Dis., 14(3): 132-140. Waskar, V.S., Zende, R.J., and Vaidya, Sheela, M.M., and Muthukmar, M. 2011. V.M. 2003. Prevalence of Listeria spp. Effectiveness of Ozone to Inactivate the in mutton and chevon. J. Vet. Pub. Listeria monocytogenes from the Milk Health., 1:65-68. samples. World J. Young Researchers., Weidmann, M., Barany, F., and Batt, C.A. 1(3): 40 – 44. 1993. Detection of Listeria United States Department of Agriculture – monocytogenes with a nonisotopic Food Safety and Inspection Service polymerase chain reaction-coupled (FSIS) (2002). Isolation and ligase chain assay. Appl. Environ. Identification of Listeria Microbiol., 59(8): 2743-2745.

How to cite this article:

Papia Biswas, Devajani Deka, T.K. Dutta, E. Motina and Roychoudhury, P. 2018. Conventional and Molecular Detection of Listeria monocytogenes and its Antibiotic Sensitivity Profile from Cattle Sources of Aizawl, Mizoram (India). Int.J.Curr.Microbiol.App.Sci. 7(11): 2829-2843. doi: https://doi.org/10.20546/ijcmas.2018.711.326

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